Reversible lysine acetylation is a highly regulated post-translational protein modification, which is controlled by protein deacetylases and acetyltransferases (Han and Martinage, 1992, Int J Biochem 24:19-28; Yang, 2004, Bioessays 26:1076-87). While originally linked to transcription and chromatin dynamics, reversible lysine acetylation is emerging as a regulator of cellular functions such as cell motility, immune synapse formation, programmed cell death, and protein trafficking (Hubbert et al., 2002, Nature 417:455-458; Kawaguchi et al., 2003, Cell 115:727-38; Serrador et al., 2004, Immunity 20:417-28; Cohen et al., 2004, Science 305:390-2; Cohen et al., 2004, Mol Cell 13:627-38). While the importance of reversible lysine acetylation of nuclear non-histone and histone proteins is well established, the role of protein modification by reversible lysine acetylation in mitochondria is unknown. However, the importance of post-translational modification of mitochondrial proteins such as phosphorylation and ADP-ribosylation, is becoming increasingly clear, and it is likely that reversible acetylation of mitochondrial proteins also plays an important role in the regulation of mitochondrial functions.
The nicotinamide (NAM) adenine dinucleotide NAD+-dependent deacetylase silent information regulator 2 (Sir2) is an important mediator of longevity in response to caloric restriction (CR) signals in Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster (Kaeberlein et al., 1999, Genes Dev 13:2570-80; Tissenbaum and Guarente, 2001, Nature 410:227-30; Lin et al., 2000, Science 289:2126-8; Rogina and Helfand, 2004, Proc Natl Acad Sci USA 101:15998-6003; Lin et al., 2004, Genes Dev 18:12-6; Lin et al., 2002, Nature 418:344-8; Anderson et al., 2003, Nature 423:181-5). Seven mammalian Sir2 homologs (SIRT1-7) are known (Frye, 1999, Biochem Biophys Res Commun 260:273-9; Frye, 2000 Biochem Biophys Res Commun 273:793-8; Blander and Guarente, 2004, Annu Rev Biochem 73:417-35; North and Verdin, 2004, Genome Biol 5:224). The recent discovery that SIRT3, SIRT4 and SIRT5 are found in mitochondria (Shi et al., 2005, J Biol Chem 280:13560-7; Michishita et al., 2005, Mol Biol Cell 16:4623-35; Onyango et al., 2002, Proc Natl Acad Sci USA 99, 13653-8; Schwer et al., 2002, J Cell Biol 158:647-57) suggests the existence of mitochondrial sirtuin substrate proteins.
The present invention discloses the identification of the first cellular acetylated substrate protein of SIRT3, Acetyl-CoA synthetase 2 (AceCS2), which is a mitochondrial matrix protein.
AceCS catalyzes the ligation of acetate with CoA to produce acetyl-CoA, an essential molecule utilized in various metabolic pathways including fatty acid and cholesterol synthesis and the tricarboxylic acid cycle (for review, see, Bremer and Osmundsen, 1984, in Fatty Acid Metabolism and Its Regulation (Numa, S. ed), 113-154, Elsevier Science Publisher, Amsterdam).
AceCS from various microorganisms and higher organisms indicates a superfamily (Toh, 1990, Protein Sequences Data Anal 3:517-521; Toh, 1991, Protein Sequences Data Anal 4:111-117), including the mammalian long chain acyl-CoA synthetases, ACS1-ACS5 (Fujino and Yamamoto, 1992, J Biochem 111:197-203; Fujino et al., J Biol Chem 271:16748-16752; Kang et al., 1997, Proc Natl. Acad Sci USA 94:2880-2884; Suzuki et al., 1990, J Biol Chem 265:8681-8685; Oikawa et al., 1998, J Biochem 124:679-685). All enzymes in this superfamily contain a common sequence motif of Ser-Gly-(small hydrophilic residue)2-Gly-(any residue)-Pro-Lys-Gly (SEQ ID NO:1) and catalyze common two-step reactions: adenylation of substrates and subsequent thioester formation (Toh, 1990, Protein Sequences Data Anal 3:517-521; Toh, 1991, Protein Sequences Data Anal 4:111-117).
Recently bovine and murine AceCS cDNAs were cloned and characterized. Two functionally distinct murine AceCSs were described: a cardiac AceCS and a hepatic AceCS. The hepatic type enzyme, termed AceCS1, is a cytosolic enzyme, whereas the cardiac enzyme, termed AceCS2, is located in the mitochondrial matrix (Fujino et al., 2001, J Biol Chem 276:11420-11426). Based on the finding that AceCS2 mRNA is induced after fasting, it was suggested that AceCS2 provides acetyl-CoA that is utilized mainly for oxidation under ketogenic conditions, such as starvation and diabetes (Fujino et al., 2001, J Biol Chem 276:11420-11426). This suggestion is supported by the finding that the level of AceCS2 mRNA in Zucker diabetic rats is increased (Fujino et al., 2001, J Biol Chem 276:11420-11426). While Fujino et al. described the induction of AceCS2 under certain conditions, they did not investigate the precise function and regulation of AceCS2 (Fujino et al., 2001, J Biol Chem 276:11420-11426).
In order to better understand and treat pathological conditions, such as ketogenic conditions, characterized by elevated levels of acetate, a substrate for AceCS2, it is important to elucidate these regulatory mechanisms. The present invention provides one such regulatory mechanism by identifying Acetyl-CoA synthetase 2 (AceCS2) as a mitochondrial enzyme and as a cellular acetylated substrate of the mitochondrial sirtuin SIRT3. AceCS2 is reversibly acetylated at lysine 642 (Lys642) in the active site of the enzyme. SIRT3 interacts with AceCS2 and deacetylates Lys642 both in vitro and in vivo. Deacetylation of AceCS2 by SIRT3 activates the acetyl-CoA synthetase activity of AceCS2.
It would be advantageous to identify agents that activate a level or deacetylase activity of SIRT3 or agents that modulate a level, acetylation status, or activity of AceCS2. Such agents would have therapeutic utility in treating pathological conditions characterized by elevated levels of acetate and other diseases or disorders that are at least partially caused by such elevated acetate levels. Here, the present invention provides methods for the identification of such agents, compositions comprising such agents and methods using such agents for the treatment of type II diabetes, hypercholesterolemia, hyperlipidemia, and obesity.